DESCRIPTION: Zinc metalloenzymes play essential roles in regulation and catalysis of metabolic pathways in all cells. The objectives of this research are to elucidate the active site structures, the function of the bound metal ion, and detailed catalytic mechanism of three classes of biologically important zinc enzymes. The enzymes to be studied are carbonic anhydrase, protein farnesyltransferase, protein geranylgeranyltransferase type I, and UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase. Inhibition of these enzymes has the potential to be used clinically for the treatment of glaucoma, diabetes, cancer, and/or Gram-negative bacterial infections. The proposed studies will substantially enhance our understanding of basic principles of these enzymes. Studies of carbonic anhydrase will use site-directed mutagenesis, followed by in-depth characterization of the variant enzymes, to investigate the thermodynamics of metal affinity and features of the transition state for CO2 hydration, including: electrophilic activation of CO2 by zinc and stabilization by hydrogen bonds. Additionally, we will select second site revertants of inactive variants to gain insight into adjustments that are required to obtain high catalytic activity in variants where the charge and structure of the active site is altered. Spectral data suggest that the catalytic role of the zinc in protein prenyltransferases is to activate a thiol for nucleophilic attack, which is a novel mechanism. We propose to further investigate the catalytic mechanism of both farnesyltransferase and geranylgeranyltransferase type I by elucidating the kinetic and spectral properties of Co(II)- and Cd(II)-substituted enzymes using absorption spectroscopy, transient kinetics, binding studies and substrate analogs in conjunction with structural studies. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase is a zinc enzyme that is the first committed step and point of regulation in the biosynthesis of lipid A in Gram-negative bacteria. We propose to elucidate the functional role of the metal in this enzyme and the catalytic mechanism by: measuring the metal specificity of the enzyme; determining spectral properties of metal-substituted deacetylase; identifying essential active site residues by site-directed mutagenesis; measuring the pH dependence of the catalytic step; and preparing deacetylase for structural studies (X-ray crystallography and/or NMR spectroscopy).